The present invention relates first, to the identification of a retrovirus and the novel nucleotide sequences encoding a retroviral polymerase gene (POL nucleotides) associated with the existence of primary sclerosing cholangitis (PSC), autoimmune hepatitis (AIH) Crohn""s disease and ulcerative colitis. The present invention further relates to methods for using the PSC associated retroviral nucleotides for the detection of PSC, AIH, Crohn""s disease and ulcerative colitis in patient samples. The present invention also relates to methods for using and targeting the PSC associated retroviral POL nucleotides in gene therapy protocols for the treatment of PSC, AIH, Crohn""s disease or ulcerative disease in patients in need of such treatment. The present invention further relates to diagnostic protocols and kits for the detection of PSC, AIH, Crohn""s disease and ulcerative colitis in tissue samples.
Extraintestinal complications of inflammatory bowel disease, including diseases of the liver and biliary tract, affect a large number of patients with ulcerative colitis Crohn""s disease. Indeed, hepatobiliary complications may affect as many as 10 percent of patients with inflammatory bowel disease. The occurrences of specific hepatobiliary diseases associated with inflammatory bowel disease, however, vary widely; some disorders occur commonly and others rarely. The clinical significances of individual hepatobiliary disorders also vary, ranging from inconsequential to life-threatening. Current data indicate that primary sclerosing cholangitis (PSC) is the most common disease associated with either ulcerative colitis or Crohn""s disease. (Harmatz A., Hepatobiliary Manifestations of Inflammatory Bowel Disease. Med. Clin. North AM 78:1387, 1994).
Primary sclerosing cholangitis (PSC) was originally defined as a chronic cholestatic liver disease characterized by fibrosing inflammation of segments of the extrahepatic bile ducts. PSC results in a progressive narrowing or obliteration of bile duct lumens, progression to secondary biliary cirrhosis, with complications of portal hypertension, hepatic failure and cholangiocarcinoma is an idiopathic disorder characterized by inflammation and obliteration of both intra-hepatic and extrahepatic bile ducts. Cholangiocytes account for 3%-5% of the hepatic cell population and line a complex network of interconnecting conduits in the liver, termed the intrahepatic biliary ductal system. One of the pathological conditions manifested in both the intrahepatic and extrahepatic bile ducts is PSC.
The natural history of PSC is incompletely understood and the variability in the location of sclerotic lesions results in a wide spectrum of clinical disease. This is especially true for asymptomatic patients and the subgroup of patients with disease confined to the intrahepatic ducts. Although as many as 30 percent of patients with the disease have demonstrable autoantibodies, there is no serologic marker associated with PSC and the diagnosis is made by radiographic demonstration of irregular strictures and dialations within the bile ducts (Vierling, Hepatobiliary complications of ulcerative colitis and Crohn""s disease. Third Edition. Philadelphia: W. B. Saunders, 1996). Patients invariably develop cirrhosis, the sequelae of liver failure, and 5 to 15 percent succumb to cholangiocarcinoma. At present, there is no curative medical therapy for PSC and liver transplantation is required to avoid these sequelae of endstage liver disease (Vierling, Hepatobiliary complications of ulcerative colitis and Crohn""s disease. Third Edition. Philadelphia: W. B. Saunders, 1996).
In North America approximately 1 in 20,000 suffer from PSC and the majority of these patients have macroscopic or microscopic evidence of either ulcerative colitis or Crohn""s disease (Vierling, Hepatobiliary complications of ulcerative colitis and Crohn""s disease. Third Edition. Philadelphia: W. B. Saunders, 1996). Other hepatic disorders may occur in patients with inflammatory bowel disease in the absence of cholangiographic evidence of large bile duct disease, including autoimmune hepatitis (AIH) and pericholangitis, a non-specific histologic diagnosis of small duct PSC. (Vierling. Hepatobiliary complications of ulcerative colitis and Crohn""s disease. Third Edition. Philadelphia: W. B. Saunders, 1996). AIH, pericholangitis, and PSC form the spectrum of the same hepatobiliary disorder associated with inflammatory bowel disease that may share a common etiological agent.
Autoimmune Hepatitis
An additional disease to which the present invention is directed is autoimmune hepatitis (AIH). Because there is no single diagnostic test for autoimmune hepatitis, it may initially be indistinguishable from other liver disorders. Early recognition is important, however, since patients suffering from autoimmune hepatitis benefit from treatment with immunosuppressives but not from treatment with interferons, used in the treatment of viral-induced hepatitis. Thus, the differentiation between viral-induced hepatitis and autoimmune hepatitis is important to ensure correct treatment. Autoimmune hepatitis, in particular in children, is an inflammatory disease which progresses into cirrhosis and hepatic insufficiency, which generally responds to an immunosuppressive treatment and is characterized by the presence of high non-organ-specific autoantibody titres.
Two subgroups have been defined, as a function of the autoantibody present in the serum: anti-smooth muscle antibody (anti-SMA) and anti-liver-kidney-microsome antibodies (anti-LKM), called hereinafter anti-SMA and anti-LKM antibodies.
The antigen recognized by the anti-LKM antibodies is a protein with a molecular weight of 50 kDa, which is present at a relatively high concentration in the endoplasmic reticulum. Several studies suggest that this antigen corresponds to a protein of the cytochrome P450 family (Waxman, 1988, Gastroenterol., 95: 1326). This was confirmed by screening a rat liver cDNA library in the presence of an anti-50 kDa protein antibody, purified by affinity from an LKM-positive serum. Both constitutive forms of cytochrome P450, belonging to the subfamily IID 1 and 2 (rat db1 and db2), have been identified, as the antigens recognized by the anti-LKM antibody (Gueguen et al., 1988 Exp. Med., 168: 801).
Other studies (Gueguen et al., 1989, Biochem. Biophys. Res. Commun, 159: 542; Zanger et al., 1988., Proc. Natl. Acad. USA, 1988, 27: 8256; Manns et al., 1989 J. Clin. Invest, 83:, 1066) have shown that cytochrome P450 IID6 is a protein recognized in human liver by the anti-LKM antibody. (Gueguen et al., 1989, Biochem. Biophys. Res. Commun, 159: 542; Zanger et al., 1988., Proc. Nati. Acad. USA, 1988, 27: 8256; Manns et al., 1989 J. Clin. Invest., 83:, 1066). Recently a solid-phase immnunoassay for the detection and quantification of LKM autoantibodies has been described (U.S. Pat. No. 5,741,654, Michel et al.) Other tests have therefore been proposed for the detection of auto-antibodies, in particular an RIA test (See Clin. Exp. Immunol., 1984 (57); 600-608. Liver-kidney microsomal auto-antibodies are detected by radioimmunoassay and their relation to anti-mitochondial antibodies in inflammatory liver diseases), which h the general disadvantages of RIA tests (especially difficulty of obtaining and handling labelled reagents).
A diagnostic test for AIH has been described which utilizes a fusion protein comprising cytochrome P450 (Gueguan et al., 1989, Biochem. Biophys. Res. Comm., 159 (2), 542-547). A human cytochrome P450 peptide fragment containing an imnmunodominant epitope of cytochrome P450 IID6 binds specifically with the anti-LKM autoantibodies produced during autoimmune hepatitis, and this peptide is the basis for a diagnostic test described in U.S. Pat. No. 5,830,667 issued to F. Alvarez, Nov. 3, 1998. Such an antigen, however, has the disadvantage of also bringing about false positives, especially because of the presence of the associated protein. Thus, one of the greatest difficulties facing those patients suffering from the spectrum of hepatobiliary disorders ranging from PSC to AIH, is not only the lack of curative medical therapy, but is the uncertainty of the curently available methods of clinically diagnosing these disorders.
The pathogenesis of PSC and AIH is probably multi-factorial in nature. There are recorded familial clusters of PSC and specific immunogenetic associations with the extended haplotype Al B8 DR3, as well as DR2 and DR52a An infectious etiology is also likely as patients with inherited and acquired immunodeficiency syndromes may also present with an identical choloangiographic appearance of strictures and dilatations within the bile ducts. Recently, evidence for a viral involvement in the pathogenesis of disease has been suggested by the finding that a proportion of patients with PSC have antibody reactivity to retroviral proteins. The importance of this finding has been reinforced by preliminary studies describing the potential involvement of a novel human retrovirus in patients with another idiopathic biliary disease, primary biliary cirrhosis.
The present invention relates, first, to the discovery, identification, and characterization of novel nucleic acid molecules, that are associated with PSC, AIH Crohn""s disease and ulcerative colitis. The novel nucleotide sequences of the present invention are retroviral in origin and, are indicative of a PSC associated retrovirus which bears a strong correlation with PSC, AIH, Crohn""s disease and ulcerative colitis.
The present inventions encompasses nucleic acid molecules which comprise the following nucleotide sequences: (a) nucleotide sequences comprising the PSC associated retroviral sequences disclosed herein; and (b) nucleotide sequences that encompass portions or fragments of the PSC associated retroviral nucleotides which can be utilized as probes or primers in the methods of the invention for identifying and diagnosing individuals at a risk for, or exhibiting PSC, AIH, Crohn""s disease or ulcerative colitis.
The invention also encompasses the expression products of the nucleic acids molecules listed above; i.e., proteins and/or polypeptides that are encoded by the above PSC associated retroviral nucleic acid molecules, or by degenerative, e.g., allelic variants thereof.
The compositions of the present invention further encompass antagonists of the PSC associated retroviral gene products, including small molecules, large molecules, and antibodies, as well as nucleotide sequences that can be used to inhibit PSC associated retroviral gene expression, e.g., antisense, ribozyme molecules, and gene or regulatory sequence replacement constructs.
The present invention relates to therapeutic methods and compositions for treatment and prevention of diseases and disorders associated with the presence of the PSC associated retroviral nucleotides, including but not limited to, PSC, AIH, ulcerative colitis and Crohn""s disease. The therapeutic methods and compositions of the present invention are designed to target the PSC associated retroviral nucleotides, such as antisense molecules and ribozymes. The therapeutic methods and compositions of the present invention are also designed to target PSC associated retroviral gene products, including small molecules, large molecules, and antibodies. The present invention further relates to the vaccine formulations based on isolated PSC associated virus particles in an attenuated form and/or PSC associated retroviral gene products for the treatment and/or prevention of disorders associated with the presence of PSC associated retroviral nucleotides.
In addition, the present invention is directed to methods that utilize the nucleotide sequences of the present invention for the diagnostic evaluation, genetic testing and prognosis of PSC associated retroviral infection and/or associated disorders including, (but not limited to PSC, AIH, ulcerative colitis and Crohn""s disease. For example, in one embodiment, the invention relates to methods of diagnosing PSC associated retroviral infection and/or associated disorders PSC, AIH, ulcerative colitis and Crohn""s disease, wherein such methods comprise measuring PSC associated retroviral gene expression in a patient sample suspected of exhibiting such a disorder. In one embodiment, nucleic acid molecules of the present invention can be used as primers for diagnostic PCR analysis for the identification of PSC associated retroviral nucleotides which correlate with the presence of a PSC associated retrovirus and/or associated disorders PSC, AIH, ulcerative colitis and Crohn""s disease. In yet another embodiment, nucleic acid molecules of the present invention can be used as primers for therapeutic PCR analysis in order to monitor the presence of a PSC associated retrovirus in order to determine the effectiveness of a therapeutic protocol.
xe2x80x9cComplementxe2x80x9d or xe2x80x9ctag complementxe2x80x9d as used herein in reference to oligonucleotide tags refers to an oligonucleotide to which a oligonucleotide tag specifically hybridizes to form a perfectly matched duplex or triplex. In embodiments where specific hybridization results in a triplex, the oligonucleotide tag may be selected to be either double stranded or single stranded. Thus, where triplexes are formed, the term xe2x80x9ccomplementxe2x80x9d is meant to encompass either a double stranded complement of a single stranded oligonucleotide-tag or a single stranded complement of a double stranded oligonucleotide tag.
The term xe2x80x9coligonucleotidexe2x80x9d as used herein includes linear oligomers of natural or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, a-anomeric forms thereof, peptide nucleic acids (PNAs), and the like, that are capable of specifically binding to a target polynucleotide. The specific binding is determined by way of a regular pattern of monomer-to-monomer interactions,such as Watson-Crick type of base pairing, base stacking, Hoogsteen or reverse Hoogsteen types of base pairing, or the like. Usually monomers are linked by phosphodiester bonds or analogs thereof to form oligonucleotides that range in size from a few monomeric units, e.g. 3-4, to several tens of monomeric units. Whenever an oligonucleotide is represented by a sequence of letters, such as xe2x80x9cATGCCTG,xe2x80x9d it will be understood that the nucleotides are in 5xe2x80x2 greater than 3xe2x80x2 order from left to right and that xe2x80x9cAxe2x80x9d denotes deoxyadenosine, xe2x80x9cCxe2x80x9d denotes deoxycytidine, xe2x80x9cGxe2x80x9d denotes deoxyguanosine, and xe2x80x9cTxe2x80x9d denotes thymidine, unless otherwise noted. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoranilidate phosphoramidate, and the like. It is clear to those skilled in the art when oligonucleotides having natural or non-natural nucleotides may be employed, e.g. where processing by enzymes is called for, usually oligonucleotides consisting of natural nucleotides are required. xe2x80x9cPerfectly matchedxe2x80x9d in reference to a duplex means that:the poly- or oligonucleotide strands making up the duplex form a double stranded structure with one other such that every nucleotide in each strand undergoes Watson-Crick basepairing with a nucleotide in the other strand. The term also comprehends the pairing of nucleoside analogs, such as deoxyinosine, nucleosides with 2-aminopurine bases, and the like, that may be employed In reference to a triplex, the term means that the triplex consists of a perfectly matched duplex and a third strand in which every nucleotide undergoes Hoogsteen or reverse Hoogsteen association with a basepair of the perfectly matched duplex. Conversely, a xe2x80x9cmismatchxe2x80x9d in a duplex between a tag and an oligonucleotide means that a pair or triplet of nucleotides in the duplex or triplex fails to undergo Watson-Crick and/or Hoogsteen and/or reverse Hoogsteen bonding. It also includes known types of modifications, for example, labels which are known in the art, methylation, xe2x80x9ccapsxe2x80x9d, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example proteins (including for e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide.
As used herein, xe2x80x9cnucleosidexe2x80x9d includes the natural nucleosides, including 2xe2x80x2-deoxy and 2xe2x80x2-hydroxyl forms, e.g. as described in Komberg and Baker, DNA Replication, 2nd Ed (Freeman, San Francisco, 1992). xe2x80x9cAnalogsxe2x80x9d in reference to nucleosides includes synthetic nucleosides having modified base moieties and/or modified sugar moieties, e.g. described by Scheit, Nucleotide Analogs (John Wiley, New York, 1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990), or the like, with the only proviso that they are capable of hybridization. Such analogs include synthetic nucleosides designed to enhance binding properties, reduce degeneracy, increase or decrease specificity, and the like.
As used herein, a polynucleotide xe2x80x9cderived fromxe2x80x9d a designated sequence refers to a subset of the designated sequence of approximately at least about 6 nucleotides, preferably at least about 8 nucleotides, more preferably at least about 1-112 nucleotides, and even more preferably at least about 15-20 nucleotides. xe2x80x9cCorrespondingxe2x80x9d means homologous to or complementary to the designated sequence. Preferably, the sequence of the region from which the polynucleotide is derived is homologous to or complementary to a sequence which is unique to an PSC associated viral genome. More preferably, the derived sequence is homologous or complementary to a sequence that is unique to all or to a majority of PSC associated viral isolates. Whether or not a sequence is unique to the a PSC associated viral genome can be determined by techniques known to those of skill in the art. For example, the sequence can be compared to sequences in databanks, e.g., Genebank, to determine whether it is present in the uninfected host or other organisms. The sequence can also be compared to the known sequences of other viral agents, including retroviruses. The correspondence or non-correspondence of the derived sequence to other sequences can also be determined by hybridization under the appropriate stringency conditions. Hybridization techniques for determining the complementarity of nucleic acid sequences are known in the art, and are discussed infra. See also, for example, Maniatis et al. (1982). In addition, mismatches of duplex polynucleotides formed by hybridization can be determined by known techniques, including for example, digestion with a nuclease such as S1 that specifically digests single-stranded areas in duplex polynucleotides. Regions from which typical DNA sequences, may be xe2x80x9cderivedxe2x80x9d include but are not limited to, for example, regions encoding specific epitopes, as well as non-transcribed and/or non-translated regions.
The derived polynucleotide is not necessarily physically derived from the nucleotide sequence shown, but may be generated in any manner, including for example, chemical synthesis or DNA replication or reverse transcription or transcription. In addition, combinations of regions corresponding to that of the designated sequence may be modified in ways known in the art to be consistent with an intended use.
The term xe2x80x9crecombinant polynucleotidexe2x80x9d as used herein intends a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin. The term further intends that the polynucleotide (1) is not associated with all or a portion of a polynucleotide with which it is associated in nature; (2) is linked to a polynucleotide other than that to which it is linked in nature; or (3) does not occur in nature.
As used herein, the xe2x80x9csense strandxe2x80x9d of a nucleic acid contains the sequence that has sequence homology to that of mRNA The xe2x80x9canti-sense strandxe2x80x9d contains a sequence which is complementary to that of the xe2x80x9csense strandxe2x80x9d.
The term xe2x80x9cprimerxe2x80x9d as used herein refers to an oligomer which is capable of acting as a point of initiation of synthesis of a polynucleotide strand when placed under appropriate conditions. The primer will be completely or substantially complementary to a region of the polynucleotide strand to be copied. Thus, under conditions conducive to hybridization, the primer will anneal to the complementary region of the analyte strand. Upon addition of suitable reactants, (e.g., a polymerase, nucleotide triphosphates, and the like), the primer is extended by the polymerizing agent to form a copy of the analyte strand. The primer may be single-stranded, or alternatively may be partially or fully double-stranded.
The terms xe2x80x9canalyte polynucleotidexe2x80x9d and xe2x80x9canalyte strandxe2x80x9d refer to a single- or double-stranded nucleic acid molecule which is suspected of containing a target sequence, and which may be present in a biological sample. As used herein, the term xe2x80x9coligomerxe2x80x9d refers-to primers and to probes. The term oligomer does not connote the size of the molecule.
As used herein, the term xe2x80x9cprobexe2x80x9d refers to a structure comprised of a polynucleotide which forms a hybrid structure with a target sequence, due to complementarity of at least one sequence in the probe with a sequence in the target region. The polynucleotide regions of probes may be composed of DNA, and/or RNA, and/or synthetic nucleotide analogs. Included within probes are xe2x80x9ccapture probesxe2x80x9d and xe2x80x9clabel probesxe2x80x9d. Preferably the probe does not contain a sequence complementary to sequence(s) used to prime the polymerase chain reaction (PCR).
As used herein, the term xe2x80x9ctarget regionxe2x80x9d refers to a region of the nucleic acid which is to be amplified and/or detected. The term xe2x80x9ctarget sequencexe2x80x9d refers to a sequence with which a probe or primer will form a stable hybrid under desired conditions.
As used herein, the term xe2x80x9cviral RNAxe2x80x9d, which includes PSC associated RNA, refers to RNA from the viral genome, fragments thereof, transcripts thereof, and mutant sequences derived therefrom.
As used herein, a xe2x80x9cbiological samplexe2x80x9d refers to a sample of tissue or fluid isolated from an individual. Thus, xe2x80x9cbiological samplexe2x80x9d, includes but is not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs, and also samples of in vitro cell culture constituents (including but not limited to conditioned medium resulting from the growth of cells in cell culture medium, putatively virally infected cells, recombinant cells, and cell components).